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Previous observations of vector-free transmission between pigs indicates the possibility of single-host cycling in swine. Therefore, the aim of this work was to investigate the evolutionary pressure of single host cycling using a relevant primary cell culture model.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      To investigate whether such single-host cycles affect viral infectivity, fitness and genomic adaptations, two strains and a reverse genetic cDNA-derived clone of JEV were serially passaged 12 times in primary porcine monocyte-derived macrophages (MDMs), in\n                      <jats:italic>Aedes albopictus<\/jats:italic>\n                      -derived C6\/36 cells, and alternately between both cell types. Next-generation sequencing analysis was used to identify selected single nucleotide variants (SNVs) and haplotypes. Phenotype-to-genotype connections were confirmed using reverse genetics.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>For all viruses, serial passaging in MDMs - but not in C6\/36 cells - led to a rapid increase in relative infectivity toward MDMs, accompanied by reduced plaque sizes in porcine endothelial cells. In contrast to C6\/36 cells, MDM imposed a strong selective pressure, rapidly favoring selection of many SNVs and viral haplotypes. In addition, we identified a dominant selection of mutants with glutamic acid to lysine substitutions at positions 49 or 138 in the E protein, which explained the small plaque phenotype and caused viral sensitivity to heparin-mediated inhibition of attachment, indicating enhanced virus binding to glycosaminoglycans (GAG). The E138K mutant also explained the increased relative infectivity for MDM.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      This work demonstrates a high evolutionary pressure on JEV in MDM causing rapid selections of minor haplotypes. Furthermore, the efficient selection of E49K and E138K SNV, which were responsible for the phenotype, are likely caused by a selective pressure for GAG binding, observed\n                      <jats:italic>in vitro<\/jats:italic>\n                      with other mammalian cells.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12985-026-03231-0","type":"journal-article","created":{"date-parts":[[2026,6,27]],"date-time":"2026-06-27T14:35:57Z","timestamp":1782570957000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Comparative dynamics of Japanese encephalitis virus adaptation in porcine macrophages and insect cells"],"prefix":"10.1186","volume":"23","author":[{"given":"Andrea","family":"Marti","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Nater","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jenny Pego","family":"Magalhaes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noelle","family":"Donz\u00e9","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Markus","family":"Gerber","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lea","family":"Almeida","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthias","family":"Liniger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra","family":"Chenaux","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicolas","family":"Ruggli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fadi G.","family":"Alnaji","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Vignuzzi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Obdulio","family":"Garcia-Nicolas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8292-4634","authenticated-orcid":false,"given":"Artur","family":"Summerfield","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,27]]},"reference":[{"key":"3231_CR1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0004139","volume":"9","author":"D Acharya","year":"2015","unstructured":"Acharya D, Paul AM, Anderson JF, Huang F, Bai F. 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